Demembranated euryhaline tilapia Oreochromis mossambicus sperm were reactivated in the presence of concentrations in excess of 10−6 M Ca2+. Motility features changed when Ca2+ concentrations were increased from 10−6 to 10−5 M. Although the beat frequency did not increase, the shear angle and wave amplitude of flagellar beating increased, suggesting that the sliding velocity of microtubules in the axoneme, which represents dynein activity, rises with an increase in Ca2+. Thus, it is possible that Ca2+ binds to flagellar proteins to activate flagellar motility as a result of the enhanced dynein activity. One Ca2+-binding protein (18 kDa, pI 4.0), calmodulin (CaM), was detected by 45Ca overlay assay and immunologically. A CaM antagonist, W-7, suppressed the reactivation ratio and swimming speed, suggesting that the 18 kDa Ca2+-binding protein is CaM and that CaM regulates flagellar motility. CaMKIV was detected immunologically as a single 48 kDa band in both the fraction of low ion extract of the axoneme and the remnant of the axoneme, suggesting that CaMKIV binds to distinct positions in the axoneme. It is possible that CaMKIV phosphorylates the axonemal proteins in a Ca2+/CaM-dependent manner for regulating the dynein activity. A 32P-uptake in the axoneme showed that 48, 75, 120, 200, 250, 380, and 400 kDa proteins were phosphorylated in a Ca2+/CaM kinase-dependent manner. Proteins (380 kDa) were phosphorylated in the presence of 10−5 M Ca2+. It is possible that an increase in Ca2+ induces Ca2+/CaM kinase-dependent regulation, including protein phosphorylation for activation/regulation of dynein activity in flagellar axoneme. Cell Motil. Cytoskeleton 2006. © 2006 Wiley-Liss, Inc.